The Putative Tumor Suppressor Tsc-22 is Downregulated Early in Chemically Induced Hepatocarcinogenesis and may be a Suppressor of Gadd45b

Mari Iida 1 2 3 4 Colleen H. Anna 2 4 Nicole D. Gaskin 2 4 Nigel J. Walker 0 2 Theodora R. Devereux 2 4 0 Toxicology Operations Branch, Environmental Toxicology Program, National Institute of Environmental Health Sciences, National Institute of Health , Research Triangle Park, North Carolina 27709 1 Present address: Department of Pathology, Biosafety Research Center , Foods, Drugs and Pesticides, Shizuoka 437-1213 , Japan 2 The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved 3 To whom requests for reprints should be addressed at Department of Pathology, Biosafety Research Center , Foods, Drugs and Pesticides, 582-2 Shioshinden, Iwata-shi, Shizuoka 437-1213 , Japan. Fax: 4 Laboratory of Molecular Carcinogenesis Tsc-22 is a novel tumor suppressor gene that represents a new class of transcription factors that has transcriptional repressor activity. We found Tsc-22 downregulation in livers from B6C3F1 mice following treatment for 2 weeks with carcinogenic doses of the antianxiety drug oxazepam (2500 ppm) or the peroxisome proliferator Wyeth-14,643 (500 ppm) but not with two other carcinogens such as o-nitrotoluene or methyleugenol or three noncarcinogens including p-nitrotoluene, eugenol, or acetaminophen. The expression of Tsc-22 was also repressed in B6C3F1 mouse liver tumors that were induced by several chemicals from 2-year carcinogenicity studies as well as in spontaneous liver tumors. To identify potential Tsc-22 target genes in mouse liver, we transfected small interference RNA (SiRNA) designed to inhibit Tsc-22 into murine liver BNL-CL.2 cells. We selected two potential transcriptional targets of Tsc-22, growth arrest and DNA damage-inducible gene 45 b (Gadd45b) and leucine zipper, putative tumor suppressor 2 (Lzts2) to test based on our previous complementary DNA microarray studies, showing that expression of these cancer-associated genes was increased when Tsc-22 was repressed. SiRNA treatment of BNL-CL.2 cells with Tsc-22 oligonucleotides but not nonspecific oligonucleotides decreased RNA and protein expression of Tsc-22 by 80-90%, while expression of Gadd45b gene, but not Lzts2, was increased over time after an initial decrease. Treatment of these cells with oxazepam for 48 h also resulted in decreased Tsc-22 and increased Gadd45b expression. These data provide evidence that Tsc-22 is a suppressor of Gadd45b expression, which may contribute to an early antiapoptotic response. - To investigate early mechanisms of mouse liver carcinogenesis following exposure to nongenotoxic carcinogens, we used complementary DNA (cDNA) microarray analysis previously to identify 25 or 36 genes with altered expression in livers from B6C3F1 mice treated with oxazepam for 2 weeks or 6 months, respectively (Iida et al., 2003). One interesting gene that was downregulated two to threefold at both time points was transforming growth factor-b (TGF-b)stimulated clone 22 (Tsc-22). Tsc-22 was first isolated as a TGF-binducible gene in a mouse osteoblast cell line (Shibanuma et al., 1992) and is an alternative spliced transcript of the TGF-b1inducible transcript 4 gene (Tgfb1i4, Gene acc. NM_009366). Tsc-22 is the only member of a novel family of transcription factors that contains a transcriptional repressor domain and does not have a DNA-binding domain. The mouse Tsc-22 gene product demonstrates strong evolutionary conservation with 98.5% and 100% identity compared to the human and rat proteins, respectively (Hamil and Hall, 1994; Jay et al., 1996). Kester et al. identified a TSC-22 binding protein, THG-1 (also called TILZ2), and they demonstrated that TSC-22 can homodimerize and heterodimerize with THG-1; they also showed transcriptional repressor activity by the Tsc-22THG-1 complex (Kester et al., 1999). Human TSC-22 was downregulated in salivary gland tumors as compared with normal salivary gland tissue and upregulated by the anticancer drug vesnarinone in a salivary gland carcinoma cell line (Kawamata et al., 1998; Nakashiro et al., 1998). After the salivary gland cancer cell line TYS received apoptotic stimuli, TSC-22 translocated from the cytoplasm to the nucleus and showed transcription-regulatory activity (Hino et al., 2000). In T47D mammary carcinoma cells TSC-22 was induced by progestin, an anticancer agent (Kester et al., 1997). Because TSC-22 was induced by anticancer agents, it was suggested to have tumor suppressor activity. Given that Tsc-22 is a putative tumor suppressor and transcriptional repressor, we hypothesized that genes expressing an inverse relationship to Tsc-22 may be targets of Tsc-22 action. From analysis of our previous cDNA microarray results, several genes were upregulated when Tsc-22 was suppressed in mice treated with oxazepam and Wyeth-14,643 for 2 weeks and 6 months, respectively, suggesting that these genes might be targets of Tsc-22. In the present study, expression of two of these genes, growth arrest and DNA damageinducible 45b (Gadd45b, genbank no. NM_008655), and leucine zipper, putative tumor suppressor 2 (Lzts2, genbank no. NM_145503) was measured by real-time and quantitative (RTAQ)-PCR to see if they were upregulated in liver after treatment for 2 weeks by carcinogens other than oxazepam or Wyeth-14,643 or by noncarcinogens in a pattern opposite to Tsc-22 expression. Gadd45b has been associated with both cell growth control and cellular response to DNA damage, demonstrating different cellular responses to different ligands. Gadd45b appears to prevent apoptotic cell death in response to tumor necrosis factor-a (TNF-a) (De Smaele et al., 2001), whereas it triggers apoptosis in response to TGF-b (Yoo et al., 2003). Lzts2, also called Lapser1, is a putative liver tumor suppressor gene that also may play a role in regulation of cell growth (Cabeza-Arvelaiz et al., 2001). To test the hypothesis that Tsc-22 plays a role in carcinogenesis, we first examined Tsc-22 expression and protein accumulation in liver tissues following treatment with various known carcinogens and noncarcinogens for 2 weeks, in liver tumors induced by different carcinogens, and in murine liver cell lines after treatment with TGF-b1 or oxazepam. To identify potential Tsc-22 target genes in mouse liver, we utilized small interference RNA (SiRNA) technology to inhibit Tsc-22 expression selectively in murine embryonic liver cell line, BNL-CL.2 and examined the expression of two genes, Gadd45b and Lzts2, identified in microarray experiments as being upregulated when Tsc-22 was repressed. MATERIALS AND METHODS Animals and experimental design. Six-week-old male or female B6C3F1 mice were obtained for the study as described previously (Iida et al., 2003). The mice were exposed for 2 weeks to different compounds, including the following treatment groups: oxazepam, male and female mice, 125 ppm (noncarcinogenic) and 2500 ppm (carcinogenic); o-nitrotoluene, female mice, 1250 ppm (noncarcinogenic) and 5000 ppm (carcinogen (...truncated)